A Comparison Study on the Degradation and Cytocompatibility of Mg

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A Comparison Study on the Degradation and Cytocompatibility of Mg-4Zn‑xSr Alloys in Direct Culture Aaron F. Cipriano,†,‡ Amy Sallee,† Ren-Guo Guan,§ Alan Lin,† and Huinan Liu*,†,‡,∥,⊥ †

Department of Bioengineering, ‡Materials Science & Engineering Program, ∥Stem Cell Center, and ⊥Cell, Molecular and Developmental Biology Program, University of California, Riverside, California 92521, United States § School of Materials Science and Engineering, Northeastern University, Shenyang 110819, China ABSTRACT: This article reports the behaviors of bonemarrow-derived mesenchymal stem cells (BMSCs) in the direct culture with four Mg-4Zn-xSr alloys (x = 0.15, 0.5, 1.0, 1.5 wt %), designated as ZSr41A, B, C, and D, respectively; and a systematic comparison on the degradation of the ZSr41 alloys and their biological impact in the direct culture with different cell types in their respective media. The direct culture method, in which cells are seeded directly onto the surface of the sample, was used to investigate cellular responses at the cell-biomaterial interface in vitro. The results showed that BMSCs adhered and remained viable on the surfaces of all ZSr41 alloys, but the faster degrading ZSr41A and ZSr41B alloys showed a significantly lower amount of viable BMSCs adhered to their surfaces. Moreover, BMSCs adhered to the culture plate surrounding the samples were unaffected by the solubilized degradation products from the ZSr41 alloys. The results from the comparison study showed that the in vitro degradation rates of Mg-based biomaterials in different culture systems might be mostly affected by media buffer capacity (i.e., HCO3− concentration), and to a lesser extent, D-glucose concentration. The comparison study also indicated that BMSCs were more robust than H9 human embryonic stem cells and human umbilical vein endothelial cells for screening the cytocompatibility of Mg-based biomaterials. In general, the adhesion and viability of BMSCs at the cell−material interface were inversely proportional to the alloy degradation rates. This study presented a clinically relevant in vitro culture system for screening bioresorbable alloys in direct culture, and provided valuable guidelines for determining the degradation rates of Mg-based biomaterials. KEYWORDS: biodegradable magnesium zinc strontium alloys, Mg−Zn−Sr alloys, degradation, bone marrow derived mesenchymal stem cells (BMSCs), cytocompatibility, in vitro direct culture method

1. INTRODUCTION Magnesium (Mg) alloys have attracted significant interest for biodegradable implant applications since the early 2000s.1−3 The promising potential of Mg-based bioresorbable implants heavily relies on the fact that the human body contains a large amount of Mg2+ ions and can effectively metabolize the degradation products of Mg.1−3 Moreover, mechanical and electrochemical properties of Mg alloys make them suitable for broad biomedical applications from orthopedic implants to vascular stents.1−4 Temporary biodegradable metallic implants are idealized to be superior alternatives to permanent implants, because they would eliminate the necessity for implant removal surgeries following the healing of damaged tissues. Therefore, Mg-based bioresorbable implants could potentially reduce the burden on the healthcare system and benefit patients by mitigating risks and costs.5,6 To realize the advantages of Mg for medical implant applications, however, it is critical to engineer the rate of Mg degradation in the physiological environment based on the end-goal design specifications. The design, processing, characterization, and mechanical properties of magnesium−zinc−strontium (Mg−Zn−Sr) ternary alloys, as well as their interactions with critical cells for cardiovascular © XXXX American Chemical Society

and neurovascular implant applications have been reported in our previous publications.7−10 This article is the first report on the degradation profiles and biological performance of Mg− Zn−Sr alloys in the direct culture with bone marrow derived mesenchymal stem cells (BMSCs), a physiologically relevant in vitro model for skeletal implant applications. We introduced the direct culture method with physiologically relevant cells to screen Mg-based alloys and characterize the cellular responses to the degradation of these metallic biomaterials for intended applications. The direct culture method provides a single, integrated culture system for assessing the degradation rates and cytocompatibility of Mgbased biomaterials, directly probing the cellular responses at the cell-material interface.11 The direct culture method addresses the limitations of current cytocompatibility tests which are based on the extract culture method (i.e., ISO 10993-5 and 10993-12) by taking the dynamic degradation processes of Mg alloys into account, thereby making it possible to study cell Received: November 4, 2016 Accepted: January 31, 2017

A

DOI: 10.1021/acsbiomaterials.6b00684 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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of ZSr41 alloys had a nominal thickness of 1 mm and were cut into 5 × 5 mm squares. Similarly, commercially pure Mg sheets (99.9%, asrolled, 1 mm thick, Cat# 40604; Alfa Aesar, Ward Hill, MA, USA) and nonculture treated glass slides (1 mm thick, Cat#12−544−1; Fisher Scientific, Hampton, NH, USA) were cut into 5 × 5 mm squares and used as a control and reference, respectively. Prior to cell culture experiments, all metallic samples were ground with SiC abrasive paper up to 1200 grit, ultrasonically cleaned for 15 min in separate baths of acetone and ethanol, individually weighed (M0), and disinfected under ultraviolet (UV) radiation for 4 h on each side. The glass samples were cleaned in acetone and ethanol, and disinfected under UV radiation following the same procedure. 2.2. BMSC Responses and in Vitro Degradation of ZSr41 Alloys. 2.2.1. Preparation of BMSC Culture. Following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California at Riverside (UCR), BMSCs were harvested from the marrow cavity of the femur and tibia of threeweek-old female Sprague−Dawley rat weanlings after euthanasia by CO2. Specific details regarding BMSC extraction, isolation and culture are described previously.20 Briefly, the distal and proximal ends of the bones were dissected and bone marrow was flushed out of the bone cavity and collected using DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 1% penicillin/streptomycin (P/S; Invitrogen, Grand Island, NY, USA). Hereafter, DMEM + 10% FBS + 1% P/S is referred to as DMEM. The collected cells were then filtered through a 70-μm nylon strainer to remove tissue debris and cell aggregates. The filtered BMSCs were cultured in DMEM under standard cell culture conditions (i.e., 37 °C, 5%/95% CO2/air, humidified, sterile environment) to 90−95% confluency. Subsequently, BMSCs were detached using Trypsin (Invitrogen) and passaged (up to the second passage, i.e., P2) for direct culture with ZSr41 alloys. 2.2.2. Direct Culture of BMSCs with ZSr41 Alloys. All the samples were placed in standard 12-well cell culture treated plates and rinsed with 2 mL of DMEM to calibrate the osmotic pressure. Subsequently, BMSCs (P2) were seeded directly onto the surfaces of the samples at a density of 4 × 104 cells cm−2 and incubated in 3 mL of DMEM under standard cell culture conditions for 72 h. A positive control, designated as “cells” group, consisted of BMSCs cultured only with DMEM in the wells, i.e., without any samples. DMEM alone (without cells and samples) was used as a blank reference and designated as “DMEM” group. In order to more closely mimic in vivo conditions where the circulation system regularly takes away soluble degradation products from the local site of implantation,8 the cell culture media were collected for analysis and replenished with 3 mL of fresh media at every 24 h interval. 2.2.3. Characterization of the BMSCs in Direct Contact with ZSr41 Alloys. The interface between the BMSCs and ZSr41 alloys, Mg control, and glass reference was characterized using a scanning electron microscope (SEM; Nova NanoSEM 450, FEI Co., Hillsboro, OR, USA) following the 72 h in vitro cultures. The corresponding surface elemental composition, and Mg Kα1 and C Kα1 elemental distribution maps were acquired with energy dispersive X-ray spectroscopy (EDS) using a Nova NanoSEM 450 equipped with an X-Max50 detector and AZtecEnergy software (Oxford Instruments, Abingdon, Oxfordshire, UK). In preparation for SEM and EDS, the samples were removed from the culture wells and dip-rinsed in phosphate buffered saline (PBS) to remove nonadherent cells. Adherent cells were fixed with 3% glutaraldehyde in 0.1 M potassium phosphate buffer for 1 h. After fixation, the samples were dip-rinsed again in PBS followed by a serial dehydration in increasing ethanol concentration (50%, 75%, 90%, 2 × 100%; 10 min each). The samples were then critical-point dried (Autosamdri-815, Tousimis Research Corp., Rockville, MD, USA) and sputter coated (model 108, Cressington Scientific Instruments Ltd., Watford, UK) with platinum/palladium at 20 mA for 40 s. An accelerating voltage of 20 kV was used to obtain SEM images and perform EDS analysis. 2.2.4. Evaluation of BMSC Adhesion and Morphology under Direct versus Indirect Contact Conditions. BMSC adhesion and morphology on the surface of the samples (direct contact with the

behaviors directly on the alloy surfaces. The direct culture method also eliminates unnecessary variables in media extraction and dilution (associated with the extract culture method), making it possible to compare the results with other in vitro studies in literature. In the direct culture method, the cells directly attached onto the Mg-based sample surfaces were defined as direct contact, whereas the cells attached to the culture plate surrounding the samples but not in direct contact with the samples were defined as indirect contact.11 The direct contact aspect of the direct culture method allowed us to probe the dynamic cell−biomaterial interface directly, whereas the indirect contact aspect in the same culture allowed us to compare our results with the results from extract-based culture method in literature.11 Therefore, the direct culture method, as an physiologically relevant in vitro model, is valuable for initial rapid screening of cytocompatibility and cytotoxicity of engineered biomaterials, although it is important to mention that in vivo evaluations at a later stage are still necessary for clinical translation.12−14 The first objective of this study was to utilize the direct culture method to investigate the degradation and cytocompatibility of four Mg-4Zn-xSr alloys (x = 0.15, 0.5, 1.0, 1.5 wt %; designated as ZSr41A, B, C, and D, respectively) with bone marrow derived mesenchymal stem cells (BMSCs) in vitro. The direct culture method was used to mimic in vivo physiological conditions and evaluate cell responses at the cell-biomaterial interface (direct contact) and on the culture plate (indirect contact; direct exposure to solubilized degradation products) surrounding the Mg-based biomaterial.11 BMSC was used for this in vitro study because of its important roles in early implant osseointegration and bone regeneration in vivo.15 Other researchers also supported the applicability of BMSCs for testing the cytocompatibility of Mg-based materials for orthopedic applications.16 Furthermore, it has been suggested that the degradation of Mg-based materials in Dulbecco’s modified Eagle medium (DMEM) resembled in vivo conditions more closely because of the presence of physiologically relevant ions and proteins.17−19 The second objective was to elucidate the key factors affecting the degradation of Mg-based biomaterials through a systemic comparison on the degradation of the ZSr41 alloys in the direct culture with different cell types in their respective media. To the best of our knowledge, this systematic comparison is also the first attempt to identify the capabilities and sensitivities of different cell types (BMSC, human embryonic stem cells and human umbilical vein endothelial cells) in detecting cytotoxic effects induced by the degradation of Mg-based biomaterials. The third objective was to compare the in vitro performance of the ZSr41 alloys with the published Mg−Zn−Ca alloys11 when the same culture methodology was used. The reported method could serve as a standard screening tool for Mg-based biomaterials.

2. MATERIALS AND METHODS We used the same experimental methods for studying Mg−Zn−Ca alloys previously.11 The description of methods was adapted for this study and reprinted from our previous publication,11 with permission from Elsevier. 2.1. Preparation of ZSr41 Alloy and Mg Control. The ZSr41 alloys in this study had a nominal composition of 4 wt % Zn with 0.15, 0.5, 1.0, or 1.5 wt % Sr; these alloys were designated as ZSr41A, ZSr41B, ZSr41C, and ZSr41D accordingly with increasing Sr content. Details on the metallurgical process and heat treatment used for the alloy preparation were published previously.7,8 The heat-treated sheets B

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ACS Biomaterials Science & Engineering sample) and on culture plate surrounding the respective samples (indirect contact with the sample) were evaluated after 72 h of culture using fluorescence microscopy. The BMSCs on the culture plates (indirect contact with the sample) were designated as “plate”. The samples in direct contact with BMSCs were removed from the wells and dip-rinsed in PBS to remove nonadherent cells. The corresponding wells were washed separately with PBS to remove nonadherent cells. Adherent cells, both on the sample surfaces and on the plates, were separately fixed with 4% formaldehyde (10% neutral buffered formalin; VWR, Radnor, PA, USA), stained with 4′,6diamidino-2-phenylindole dilactate (DAPI; Invitrogen) nucleic acid stain and Alexa Flour 488-phalloidin F-actin stain, and imaged using a fluorescence microscope (Eclipse Ti and NIS software, Nikon, Melville, NY, USA) with a 10x objective lens at the same exposure condition. Fluorescence images of BMSCs were analyzed using ImageJ (NIH, Bethesda, MD, USA). Cell adhesion for each group was quantified by counting the DAPI-stained cell nuclei at five random locations on the sample surface and at nine random locations on the culture plate. Cell adhesion density was calculated as the number of adherent cells per unit area. 2.2.5. Evaluation of ZSr41 Alloy Degradation in the Direct Culture with BMSCs in Vitro. At each prescribed incubation interval, the in vitro degradation of the ZSr41 alloys and Mg was evaluated through measurements of pH and ionic concentrations of the collected media. The pH of the media at each incubation interval was measured immediately after collection using a calibrated pH meter (Model SB70P, VWR). The concentration of Mg2+, Zn2+, and Sr2+ ions in the collected media was measured using inductively coupled plasma− optical emission spectrometry (ICP-OES; Optima 8000, PerkinElmer, Waltham, MA, USA). To minimize the matrix effects in ICP-OES, the collected DMEM aliquots were diluted to 1:100 solutions in DI water. Ionic concentrations were calculated based on the calibration curves generated using Mg, Zn, and Sr standards (PerkinElmer) that were diluted to the ranges of 0.5−5.0, 0.1−1.0, and 0.1−1.0 mg/L, respectively. 2.3. Statistical Analyses. BMSC culture and in vitro degradation experiments were run in triplicate. All data sets were tested for normal distribution and homogeneous variance. Parametric data sets were analyzed using one-way analysis of variance (ANOVA) followed by the Tukey HSD post hoc test. Data sets with normal distribution but heterogeneous variance were analyzed using one-way ANOVA (homogeneous variance not assumed) followed by the Games-Howell post hoc test. Nonparametric data sets were analyzed using the Kruskal−Wallis test followed by the Nemenyi post hoc test. Statistical significance was considered at p < 0.05. 3. Results. 3.1. BMSCs in Direct Contact with ZSr41 Alloys. Figure 1 shows the SEM characterization of the interface between the BMSCs and the materials of interest at an original magnification of 150× and 1000× after 72 h of direct culture; the BMSCs were in direct

contact with the samples. The ZSr41 alloys showed distinct surface microstructure and topography when compared with the Mg control and glass reference. Degradation-induced surface cracks and secondary phases (indicated with black arrows) were observed on the surfaces of all ZSr41 alloys. As expected, the presence of secondary phases increased with increasing Sr content, in agreement with our previous characterization of the ZSr41 alloys.10 All ZSr41 alloys showed similar characteristics in terms of the size and distribution of degradation cracks. In contrast, the Mg control samples showed more prevalent surface cracks and severe localized degradation. Furthermore, adhered BMSCs (indicated with white arrows) were observed on the surfaces of all ZSr41 alloys and Mg control samples. In all cases, the BMSCs showed a high degree of isotropic spreading,11,21 but the cell density on Mg-based samples was visibly lower than the glass reference on which the BMSCs showed complete confluency after 72 h culture. Qualitative and semiquantitative assessment of the elemental distribution and composition provided further evidence of changes in structural integrity and mineral deposition at the BMSC-material interface after 72 h of culture. Figure 2 shows the elemental

Figure 2. Surface composition of the ZSr41 alloys and Mg control after 72 h of direct culture with BMSCs in DMEM. (a) EDS maps of elemental distribution of Mg and C (Kα1 lines) for the ZSr41 alloys and pure Mg control. Original magnification = 150×; scale bar = 250 μm for all images. (b) Surface elemental composition (wt %) based on EDS quantification on 150× images. distribution maps of Mg and carbon (C; Kα1 lines for both) and a summary of elemental composition (in wt %) of the samples quantified through EDS analyses at 150× magnification after 72 h of culture. The Mg distribution maps (Figure 2a) showed that the surfaces of ZSr41 alloys remained more intact when compared with the Mg control, and indicated similar degradation mode for the ZSr41 alloys up to 72 h. Additionally, the C distribution maps (Figure 2a) of all metallic samples confirmed the adhesion of BMSCs (brighter regions) along with other organic components (most likely protein and extracellular matrix deposition). The stacked histogram shown in Figure 2b summarizes the elemental composition (in wt %) for the samples of interest, quantified through EDS analyses at 150× magnification. A Zn content comparable to the nominal 4% was detected on the surfaces of ZSr41A, B, C, and D alloys (3.81, 4.46, 3.04, and 4.92%, respectively); and Zn was not detected on the Mg control or glass reference. Additionally, increasing Sr content was also detected on ZSr41B, C, and D alloys (0.08, 0.34, and 0.84%, respectively), but Sr was not detected on the surface of ZSr41A alloy,

Figure 1. SEM images of BMSCs in direct contact with the ZSr41 alloys after 72 h of direct culture in DMEM. The micrographs of ZSr41 alloys, pure Mg control, and glass reference were taken at an original magnification of 150×; scale bar = 250 μm for all images. Insets were taken at an original magnification of 1000× with scale bar =50 μm for all images. Black arrows indicate secondary phases and white arrows indicate adhered and spread BMSCs. C

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ACS Biomaterials Science & Engineering Mg control, or glass reference. Interestingly, all Mg-based samples showed colocalization of calcium (Ca), phosphorus (P), and oxygen (O), which indicated that Mg degradation possibly attracted Cacontaining mineral deposition. The deposition of Ca, P, and O was similar in wt % and exclusive for all Mg-based materials, which is consistent with previously published results.10,11 In contrast, the glass reference did not show deposition of Ca and P; the content of Ca and O and the identified elements closely matched the composition of soda lime glass characterized before cell culture.11 Lastly, all samples showed sodium (Na) and potassium (K) content because of salt deposition from the culture media. 3.2. BMSC Adhesion under Direct and Indirect Contact Conditions. Figure 3 summarizes the results of BMSC adhesion

during the 72 h of incubation (in every 24 h interval) are summarized in Figure 4. Media pH and Mg2+ ion concentration ([Mg2+]) are both

Figure 4. Analysis of solubilized degradation products in culture media at 24, 48, and 72 h of direct culture with the ZSr41 alloys, pure Mg control, and glass reference, as well as the cells-only positive control, and blank DMEM media. (a) pH of media, (b) Mg2+ ion concentration, (c) Zn2+ ion concentration, and (d) Sr2+ ion concentration. Values are mean ± SD, n = 3 at all time points. *p < 0.05, **p < 0.01, ***p < 0.001. important indicators for the degradation of Mg-based materials;14 in addition the Zn2+ and Sr2+ ion concentrations ([Zn2+] and [Sr2+], respectively) were measured to ensure their concentrations remained below the reported cytotoxic levels.8,10 ANOVA was used to confirm statistically significant differences in the mean pH of the collected culture media at 24 h [F (7, 16) = 4.5647, p = 5.685 × 10−3], 48 h [F (7, 16) = 7.7447, p = 3.672 × 10−4], and 72 h [F (7, 16) = 13.21, p = 1.40 × 10−5] (Figure 4a). Specifically, post hoc pairwise comparisons at 24 h showed that the pH of the culture media with Mg (8.13 ± 0.01) was significantly higher (p < 0.05) than that for ZSr41A (8.06 ± 0.02) and the glass reference (8.06 ± 0.01). At 48 h, post hoc pairwise comparisons showed that pH of the media for glass reference (7.95 ± 0.01) and BMSC-only positive control (7.96 ± 0.02) was significantly lower (p < 0.01 and p < 0.05, respectively) than ZSr41C (8.03 ± 0.04), ZSr41D (8.04 ± 0.02), and DMEM-only blank reference (7.95 ± 0.01). At 72 h, post hoc tests showed that the pH of the culture media for ZSr41B (8.07 ± 0.05) was significantly higher (p < 0.05) than the Mg control (7.98 ± 0.03) and glass reference (7.94 ± 0.02); the pH of the media for ZSr41C (8.15 ± 0.05) was significantly higher (p < 0.05) than the ZSr41A (8.02 ± 0.05); both ZSr41C and ZSr41D (8.11 ± 0.02) were significantly higher (p < 0.001 and p < 0.05, respectively) than the Mg control, glass reference, and BMSC-only positive control (7.99 ± 0.02). Furthermore, the pH of the DMEM blank reference (8.07 ± 0.02) was significantly higher (p < 0.05) than the glass reference. The Kruskal−Wallis test for nonparametric data was used to confirm statistically significant differences in the mean [Mg2+] of the collected culture media at 24 h [X2 (7, N = 24) = 19.2267, p = 7.506 × 10−3] and 48 h [X2 (7, N = 24) = 18.8267, p = 8.748 × 10−3]. ANOVA confirmed statistically significant differences at 72 h [F (7, 16) = 33.958, p = 1.92 × 10−8] (Figure 4b). At 24 h, post hoc pairwise comparisons showed that the [Mg2+] in the culture media of ZSr41A (3.15 ± 0.73 mM) and ZSr41B (2.72 ± 0.18 mM) were significantly higher (p < 0.05) than the BMSC-only positive control (0.59 ± 0.10 mM) and DMEM blank reference (0.59 ± 0.13 mM). At 48 h, post hoc pairwise comparisons showed that the [Mg2+] in the culture media of ZSr41A (2.72 ± 0.33 mM) was significantly higher (p < 0.05) than the glass (0.60 ± 0.08 mM) and DMEM blank (0.63 ± 0.08 mM) references. Additionally, both ZSr41B (2.56 ± 0.19 mM) and ZSr41C

Figure 3. BMSC adhesion on the ZSr41 alloys, pure Mg control, glass reference, and cells-only positive control (labeled as “BMSC”) after 72 h of direct culture. (a) Representative fluorescence images of adhered BMSCs on the tissue culture plates at 72 h. (b) Adhesion density of BMSCs on the sample surface (direct contact with the sample) and on the culture plate surrounding each corresponding sample (indirect contact with the sample). Values are mean ± standard error of the means, n = 3; *p < 0.05. directly onto the sample surface (direct contact) and on the culture plate (indirect contact) after 72 h of direct culture with the ZSr41 alloys, Mg control, and glass reference. Figure 3a shows representative fluorescence images of BMSCs attached on the culture plates (indirect contact) at 72 h postincubation; similar images (not shown) were taken for the BMSCs under direct contact at 72 h. The representative fluorescence images showed that BMSCs under indirect contact grew to confluency during the 72 h culture with the samples. The DAPIstained nuclei were used to quantify cell adhesion density and the results are plotted in in Figure 3b. ANOVA was used to confirm statistically significant differences in the mean cell adhesion density on the sample surface (direct contact) [F (5, 12) = 3.985, p = 2.312 × 10−2]. Specifically, post hoc pairwise comparisons showed that the cell adhesion density on the surface of ZSr41A (0.28 ± 0.14 × 104 cells cm−2) and ZSr41B (0.13 ± 0.08 × 104 cells cm−2) was significantly lower (p < 0.05) than the glass reference (2.97 ± 0.70 × 104 cells cm−2). The cell adhesion density on the culture plate (indirect contact) showed no statistically significant differences among all the samples and controls. 3.3. Degradation Rates of ZSr41 Alloys in the Direct Culture with BSMCs in vitro. The analyses of the DMEM culture media D

DOI: 10.1021/acsbiomaterials.6b00684 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering (2.54 ± 0.07 mM) were significantly higher (p < 0.05) than the glass reference. At 72 h, post hoc pairwise comparisons showed that the [Mg2+] in the culture media of all ZSr41 alloys was significantly higher (p < 0.001) than the nonmetallic references. That is, ZSr41A (2.65 ± 0.40 mM), ZSr41B (2.48 ± 0.22 mM), ZSr41C (2.37 ± 0.11 mM), and ZSr41D (2.39 ± 0.19 mM) were all significantly higher than the glass reference (0.68 ± 0.05 mM), BMSC-only positive control (0.61 ± 0.06 mM), and DMEM-only blank reference (0.77 ± 0.21 mM). The Mg control (1.77 ± 0.50 mM) was significantly higher than the glass, BMSC, and DMEM groups. The only statistically significant difference (p < 0.05) among the Mg-based groups was confirmed between ZSr41A and the Mg control. In general, as expected, all of the Mg-based samples caused an increase in the [Mg2+] in the cell culture media. Figure 4c, d show [Zn2+] and [Sr2+], respectively, in the cell culture media after 72 h of incubation. No statistically significant differences were detected for [Zn2+] and [Sr2+]. In general, the [Zn2+] and [Sr2+] in the culture media were in the single-digit μM range. The degradation rates for each ZSr41 alloy and pure Mg control (given as a mass loss rate per unit area per day) are summarized in

mTeSR1 culture media8 and with human umbilical vein endothelial cells (HUVECs) in EGM-2 culture media.10 Additionally, the degradation rates of ZSr41 from this study were compared with the degradation rates of Mg-xZn-0.5Ca alloys (x = 0.5, 1.0, 2.0, and 4.0 wt %) under the same culture conditions (i.e., 72 h of direct culture with BMSCs).11 The original data from previous publications8,11 were used to calculate the degradation rates according to eq 1. It is important to mention that pure Mg controls for all four studies of degradation (and cytocompatibility) consisted of 99.9% pure, as-rolled sheets with the following thickness: 250 μm thick (Goodfellow Co.) for the ZSr41hESC/mTeSR1 study;8 1 mm thick (Alpha Aesar) for ZSr41HUVEC/EGM-2 study10 and this ZSr41-BMSC/DMEM study; and 500 μm thick (Goodfellow Co.) for Mg-xZn-0.5Ca-BMSC/DMEM study.11 The comparison for the degradation rates of the ZSr41 alloys in hESC/mTeSR1, HUVEC/EGM-2, and BMSC/DMEM, along with the comparison with Mg-xZn-0.5Ca in BMSC/DMEM are summarized in Figure 6. For ease of visualization, the results from this study

Figure 6. Comparison of daily degradation rates of (a) ZSr41 alloys in BMSC/DMEM (this study), HUVEC/EGMTM-2,10 and hESC/ mTeSR18 culture systems during 24 h of culture; and (b) ZSr41 alloys and Mg-xZn-0.5Ca (x = 0.5, 1.0, 2.0, and 4.0 wt %) alloys11 in BMSC/DMEM culture system during 72 h of direct culture. All Mg controls were 99.9% pure, as-rolled sheets with the following thickness: 250 μm thick (Goodfellow Co.) for ZSr41-hESC/ mTeSR1 study; 1 mm thick (Alpha Aesar) for ZSr41-HUVEC/ EGM-2 and ZSr41-BMSC/DMEM study; and 500 μm (Goodfellow Co.) for Mg-xZn-0.5Ca-BMSC/DMEM study. Values are mean ± SD, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Average daily degradation rate (mass loss rate) per unit surface area for the ZSr41 alloys and pure Mg during 72 h of direct culture with BMSCs in DMEM. Values are mean ± SD, n = 3; *p < 0.05. Figure 5. These values were calculated according to the following eq (eq 1): DR =

([Mg 2 +]i ,tot − [Mg 2 +]DMEM,tot )V SA it

are represented by the red striped columns in all panels. Figure 6a shows the normalized degradation rates of ZSr41 alloys and Mg control (1 mm thick; Alfa Aesar) in different cell culture media with corresponding cell types over the 24 h of culture. The Mg control group in mTeSR1 was excluded from the comparison since it was geometrically distinct from the Mg control in DMEM and EGM-2. ANOVA was used to confirm statistically significant differences in the mean degradation rates of ZSr41A [F (2, 6) = 72.715, p = 6.22 × 10−5], ZSr41C [F (2, 6) = 14.289, p = 5.23 × 10−3], and Mg [F (1, 4) = 288.92, p = 7.03 × 10−5], whereas the Kruskal−Wallis test was used to confirm differences for ZSr41B [X2 (2, N = 9) = 7.2, p = 2.732 × 10−2] and ZSr41D [X2 (2, N = 9) = 7.2, p = 2.732 × 10−2] (Figure 6a). Post hoc pairwise comparisons for ZSr41A showed that the degradation rate in BMSC/DMEM (0.36 ± 0.07 mg cm−2 day−1) was significantly lower (p < 0.01) than in HUVEC/EGM-2 (0.93 ± 0.03 mg cm−2 day−1) and hESC/mTeSR1 (0.69 ± 0.07 mg cm−2 day−1); the degradation rate of ZSr41A in hESC/mTeSR1 was significantly lower (p < 0.01) than in HUVEC/EGM-2. Similarly, post hoc pairwise comparisons for ZSr41B showed that the degradation rate in BMSC/DMEM (0.34 ± 0.06 mg cm−2 day−1) was significantly lower (p < 0.05) than in HUVEC/EGM-2 (1.63 ± 0.54 mg cm−2 day−1) and hESC/mTeSR1 (0.56 ± 0.03 mg cm−2 day−1); the degradation rate of ZSr41B in hESC/mTeSR1 was significantly lower (p < 0.05) than in HUVEC/EGM-2. Furthermore, post hoc pairwise

(1)

Where [Mg2+]i,tot represents the total [Mg2+] in the culture media (in mg/L) from all three 24-h incubation periods for each sample in each group “i″ (i = ZSr41A, B, C, D, and Mg), [Mg2+]DMEM,tot represents the total [Mg2+] in the culture media from all three 24-hr incubation periods for the DMEM-only reference (i.e., [Mg2+] in DMEM for correction), V represents the media incubation volume (0.003 L), SAi represents the initial surface area (cm2) of each sample in group “i” based on the initial geometry of the sample, and t is the total incubation time (in days). The degradation rate was calculated as [mg cm−2 day−1]. ANOVA confirmed statistically significant differences in the normalized degradation rates during the 72 h incubation period [F (4, 10) = 5.8336, p = 1.09 × 10−2]. Post hoc pairwise comparisons showed that the degradation rates of ZSr41A (0.308 ± 0.047 mg cm−2 day−1) and ZSr41B (0.306 ± 0.038 mg cm−2 day−1) were significantly higher (p < 0.05) when compared with the Mg control (0.174 ± 0.058 mg cm−2 day−1). 3.4. Degradation of ZSr41 Alloys in Direct Culture: A Comparison Study on the Culture Systems and Mg Alloys. The degradation rates of the ZSr41 alloys obtained from the direct culture with BMSCs in DMEM and eq 1 were compared with our previous publications in which we reported the degradation rates of ZSr41 alloys cultured with human embryonic stem cells (hESCs) in E

DOI: 10.1021/acsbiomaterials.6b00684 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering comparisons for ZSr41C showed that the degradation rate in BMSC/ DMEM (0.25 ± 0.03 mg cm−2 day−1) was significantly lower (p < 0.01) than in HUVEC/EGM-2 (0.70 ± 0.18 mg cm−2 day−1) and hESC/mTeSR1 (0.65 ± 0.07 mg cm−2 day−1). Similar to ZSr41A and ZSr41B, post hoc pairwise comparisons for ZSr41D showed that the degradation rate in BMSC/DMEM (0.27 ± 0.04 mg cm−2 day−1) was significantly lower (p < 0.05) than in HUVEC/EGM-2 (1.67 ± 0.52 mg cm−2 day−1) and hESC/mTeSR1 (0.51 ± 0.06 mg cm−2 day−1); the degradation rate of ZSr41D in hESC/mTeSR1 was significantly lower (p < 0.05) than in HUVEC/EGM-2. In addition, post hoc pairwise comparisons for Mg control showed that the degradation rate in BMSC/DMEM (0.23 ± 0.01 mg cm−2 day−1) was significantly lower (p < 0.001) than in HUVEC/EGM-2 (0.72 ± 0.05 mg cm−2 day−1). Figure 6b shows a comparison of the normalized daily degradation rates of ZSr41 alloys, Mg-xZn-0.5Ca alloys, and their respective Mg controls over the 72 h of direct culture in BMSC/DMEM system. The average degradation rates of most Mg-xZn-0.5Ca alloys were generally slower than the ZSr41 alloys. Specifically, the Mg-xZn-0.5Ca alloy with x = 2.0 wt % showed the slowest degradation rate on average among all the Mg-4Zn-xSr and Mg-xZn-0.5Ca alloys.11 3.5. Cytocompatibility of ZSr41 Alloys in Direct Culture: A Comparison Study on the Relevant Cells in Their Respective Media. Cytocompatibility results from the direct culture of ZSr41 alloys with BMSCs (this study) were compared with our previous publications in which we reported the cytocompatibility of ZSr41 alloys with hESCs8 and HUVECs,10 and the cytocompatibility of MgxZn-0.5Ca alloys with BMSCs.11 We compared different culture systems (i.e., different cell types with their respective media, hESC/ mTeSR1, HUVEC/EGM-2, BMSC/DMEM) for evaluating the cytocompatibility of the same ZSr41 alloys, and compared the cell viability with ZSr41 and Mg-xZn-0.5Ca alloys when the same culture system (i.e., BMSC/DMEM) was used. To facilitate comparison, the viability of each cell type was represented as percent cell viability and was calculated as follows: percent viability of hESCs was determined by the area of viable hESC colonies in the experimental groups and normalized by the cells-only positive control at either 24 or 72 h;8 percent viability of HUVECs10 and BMSCs (11 and this study) was determined by the adhesion density of DAPI-stained cell nuclei and normalized by the cells-only positive control at respective time point (i.e., 72 h for BMSCs and 24 h for HUVECs). It is important to mention that Transwell inserts were used to introduce the samples in the ZSr41-hESC study, thus only providing cytocompatibility results similar to indirect contact conditions. The comparison for the cytocompatibility of ZSr41 and Mg-xZn0.5Ca alloys with hESCs, HUVECs, and BMSCs are summarized in Figure 7. For ease of visualization, the results from this study are represented by the red striped columns in all panels. Figure 7a shows the percent cell viability of BMSCs and hESCs under indirect contact with ZSr41 alloys; the results showed that after 72 h of incubation with the ZSr41 alloys and Mg control, the percent viability of BMSCs was much higher than hESCs. Furthermore, Figure 7b shows the percent cell viability of hESCs and HUVECs under indirect contact with the ZSr41 alloys and Mg control; the results showed that the percent viability of HUVECs was higher than the viability of hESCs after 24 h of incubation. The percent viability of BMSCs in the direct culture under both direct and indirect contact with ZSr41 alloys and Mg-xZn0.5Ca alloys after 72 h of incubation was also plotted for comparison, as shown in Figure 7c, d, respectively. Comparison for the direct contact conditions in Figure 7c showed that, in general, the BMSCmaterial interface of the Mg-xZn-0.5Ca alloys was more favorable for the adhesion and continued viability of BMSCs than the ZSr41 alloys. In contrast, comparison for the indirect contact conditions in Figure 7d showed that BMSCs growing on the culture plate remained unaffected by the degradation rates (and concomitant solubilized degradation products) of both Mg−Zn−Sr and Mg−Zn−Ca alloy groups after 72 h of incubation.

Figure 7. Comparison of the percentages of cell viability in different culture systems with ZSr41 alloys and Mg-xZn-0.5Ca alloys. The percent cell viability was normalized by the cells-only positive control. (a) Percent cell viability for BMSCs when compared with hESCs, in their respective culture media, at 72 h under indirect contact with the ZSr41 alloys and Mg control. (b) Percent cell viability for hESCs when compared with HUVECs, in their respective culture media, at 24 h under indirect contact with the ZSr41 alloys and Mg control. (c) Percent BMSC viability with ZSr41 alloys as compared with Mg-xZn0.5Ca (x = 0.5, 1.0, 2.0, and 4.0 wt %) alloys after 72 h of direct culture under direct contact conditions. (d) Percent BMSC viability with ZSr41 alloys as compared with Mg-xZn-0.5Ca (x = 0.5, 1.0, 2.0, and 4.0 wt %) alloys after 72 h of direct culture under indirect contact conditions. Percent viability of hESCs was determined by the area of viable hESC colonies in the experimental groups and normalized by the positive control at each time point (i.e., 24 and 72 h).8 Percent viability of HUVECs [10] and BMSCs [11] was determined by the adhesion density of DAPI-stained cell nuclei and normalized by the positive control at respective time point (i.e., 24 h for HUVECs and 72 h for BMSCs). All Mg controls were 99.9% pure, as-rolled sheets with the following thickness: 250 μm thick (Goodfellow Co.) for ZSr41hESC/mTeSR1 study; 1 mm thick (Alpha Aesar) for ZSr41-HUVEC/ EGM-2 and ZSr41-BMSC/DMEM study; and 500 μm (Goodfellow Co.) for Mg-xZn-0.5Ca-BMSC/DMEM study.

4. DISCUSSION The degradation of four heat-treated ZSr41 crystalline Mg alloys and their cytocompatibility with BMSCs were investigated using the direct culture method in vitro. This direct culture method not only enabled us to study the possible modulatory effects of cells at their direct interface with Mgbased alloys, specifically ZSr41 alloys in this study, but also allowed us to examine the BMSCs on the culture plates surrounding the Mg-based samples in the same direct culture.11 The direct culture method should be further considered as a standard method for evaluating cytocompatibility of biodegradable and/or bioresorbable biomaterials in vitro, considering the benefits of simultaneously examining the behaviors of cells that are in direct contact and indirect contact with the biomaterial samples in the same in vitro culture system. Moreover, the degradation and cytocompatibility results from this study were systematically compared with published literature to elucidate the effects of the culture systems, i.e., relevant cells in their respective culture media, as well as the degradation and F

DOI: 10.1021/acsbiomaterials.6b00684 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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The systemic comparison presented in this study showed that the in vitro degradation rates of ZSr41 in BMSC/DMEM, hESC/mTeSR1, and HUVEC/EGM-2 were likely independent of the following media parameters, including chloride ion concentration ([Cl−]), osmolality, and/or ionic strength. Table 1 summarizes the ionic concentrations from the inorganic salts,

cytocompatibility of the ZSr41 alloys versus Mg-xZn-0.5Ca alloys. 4.1. In Vitro Degradation of ZSr41 Alloys in Direct Culture. The ZSr41A and ZSr41B alloys showed significantly faster degradation rates in vitro than the Mg control in this study after 72 h of direct culture with BMSCs in DMEM. The degradation rate data (Figure 5) was qualitatively supported by the more heterogeneous surface integrity of ZSr41A and ZSr41B alloys in contrast to the more homogeneous surfaces of ZSr41C and ZSr41D alloys, as indicated in the postincubation SEM images (Figure 1) and Mg Kα1 maps (Figure 2a). Interestingly, the Mg control showed the slowest degradation rate but also the most heterogeneous surface integrity among all the Mg-based samples investigated in this study. The pH values measured during the 24 h intervals (Figure 4a) showed that although the degradation of all Mg-based samples induced an alkaline shift in the culture media, the CO2/HCO3− buffer system in DMEM was effective in maintaining media pH well below pH 9.5. The media pH of 9.5 was the critical alkalinity when adverse effects for BMSCs were observed according to our previous study.11 Moreover, the results of [Mg2+] in the culture media during the 24 h intervals (Figure 4b) showed a nearly constant release of Mg2+ ions from the degradation of all ZSr41 alloys during the 72 h culture. In contrast, the mean Mg2+ ion release from the degradation of the Mg control showed a continuously decreasing trend in degradation rate during the 72 h incubation with the lowest mean value measured during the last 24-h interval. In a previous study, we demonstrated that the differences in in vitro degradation rates of the four ZSr41 alloys could be potentially correlated with specific Zn/Sr at % ratio in the β-phase of these alloys and the corresponding microgalvanic corrosion formed with the αmatrix.10 Additionally, the solubilized [Mg2+], [Zn2+], and [Sr2+] in the culture media from the degradation of the ZSr41 alloys during the prescribed 24-h intervals were all below therapeutic daily doses and LD50 concentrations for these three ions. In other culture systems reported previously,8,10 [Mg2+], [Zn2+], and [Sr2+] in the media from the degradation of the ZSr41 alloys were also below the toxic levels. The systemic comparison of the results from this study with previous publications8,10 showed that the in vitro degradation rates of ZSr41 alloys in distinct culture systems increased as follows: BMSC/DMEM < hESC/mTeSR1 < HUVEC/EGM-2. In all cases, the degradation rates of the ZSr41 alloys and Mg control in DMEM were significantly lower when compared with their corresponding counterparts in mTeSR1 and EGM-2 cultures. Furthermore, the degradation rates of ZSr41 alloys in mTeSR1 appeared to be slower than in EGM-2 in average, although the difference was not statistically significant for all the cases. Interestingly, in terms of degradation rate, the ZSr41C and ZSr41D alloys performed better in BMSC/DMEM than the ZSr41A and ZSr41B alloys during the 72 h incubation reported in this study. In contrast, ZSr41A and ZSr41C showed a slower degradation rate compared with ZSr41B and ZSr41D when incubated for 72 h in hESC/mTeSR1. The degradation of ZSr41 alloys was also studied in whole blood, blood plasma, and fibroblast culture; however, the culture parameters were different from this study and thus was not included in the comparison.9 Despite the distinct degradation rates observed in the various culture systems, the ZSr41C alloy appeared to be the candidate with consistently lower degradation rate when compared with ZSr41A, B, and D.

Table 1. Ionic Concentrations (mM) from Inorganic Salts in DMEM, MCDB 131 (non-commercial analog of EGM-2), mTeSR®1, and r-SBF in Comparison with Human Blood Plasmaa DMEM Na+ K+ Mg2+ Li2+ Ca2+ Fe3+ Cl− HCO3− H2PO4− HPO42− SO42− Ionic Strength D-glucose Osmolality (mOsm/kg H2O)b

MCDB 131

156.46 5.37 0.81

124.5 4 10

1.80 0.0002 118.48 44.04 0.0007 0.91

1.6 0.001 117.2 14 0.5

0.168 24.98 335 ± 30c

10 0.173 5.55 275 ± 15d

r-SBF

Plasma

113.74 3.26 0.56 0.98 0.82

mTeSR1

142 5 1.5

142 5 1.5

2.5

2.5

100.96 18 0.36 0.39 0.32 0.123 13.75 240 ± 10e

103 27

103 27

1 0.5 0.150

1 0.5 0.150 5 290

a

Compiled from refs 22−26. bOsmolality values reported with the presence of organic species and buffers. cOsmolality for DMEM media from technical specification sheet. Corning Cat# 50−013. dOsmolality for EGM-2 media from technical specification sheet. Lonza Cat# CC3162. eOsmolality for mTeSR1 media from technical specification sheet. Stem Cell Tech, Cat# 85850.

ionic strength, D-glucose, and osmolality of DMEM,22 MCDB 131 (noncommercial analog of EGM-2),23 mTeSR1,24 r-SBF,22 and human blood plasma.24 The D-glucose and osmolality values for DMEM, EGM-2, and mTeSR1 were obtained from technical specification sheets from the vendors, while the values for human blood plasma were obtained from refs 25 and 26, respectively. The presence of chloride ions (Cl −) in physiological fluids has been extensively argued to play a dramatic role in the degradation of Mg-based materials,1−3 especially when the [Cl−] > 30 mM.2 However, the degradation rates of ZSr41 alloys in distinct culture media with corresponding [Cl−] (i.e., DMEM = 118.48 mM, MCDB 131/EGM-2 = 117.2 mM, and mTeSR1 = 100.96 mM; Table 1) appear to be largely influenced by other factors. For example, a significantly faster degradation rate of ZSr41 was observed for culture media with lower [Cl−], i.e., mTeSR1 had lower [Cl−] but induced a faster degradation rate than DMEM. Similarly, the significantly faster degradation rate of ZSr41 alloys in EGM-2 occurred despite a midrange [Cl−] relative to the concentration in DMEM and mTeSR1. Additionally, our results showed that the significantly faster degradation rate observed for the ZSr41 alloys in EGM-2 appeared to be independent of media osmolality and ionic strength since (i) DMEM had the highest nominal media osmolality, yet induced the slowest degradation rate, and (ii) the degradation rate in DMEM was slowest despite DMEM having a midrange ionic G

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glucose, in that order, while maintaining all the other factors at a constant level. The types of cells used in the culture could also influence the degradation behaviors of Mg-based alloys in vitro, although it is difficult to isolate the cell type factor because the optimal media for culturing BMSCs, hESCs, and HUVECs vary. Recent studies have indicated that the cells, such as human primary osteoblasts,30 SaOS-2 cells (primary osteogenic sarcoma),31 murine L929 fibroblasts,32 and RAW264.7 macrophages,33 affected the degradation behaviors of Mg-based alloys. For example, it was reported that human primary osteoblasts altered the degradation interface actively with their metabolic activity and passively by their adhesion and cell layer formation.30 Witecka et al. attributed the accelerated corrosion of as-cast Mg-2.0Zn-0.98Mn (ZM21) alloy to the decrease of medium pH due to cellular metabolic activities when SaOS-2 cells were cultured on the ZM21 alloy samples.31 Further experiments with the direct culture method are necessary to elucidate the different roles of BMSC, hESC, and HUVEC at the cell− material interface on the degradation rates of Mg-based biomaterials. In terms of the degradation performance of Mg alloys, our comparison study showed that the degradation rates of the MgxZn-0.5Ca alloys were slower than the ZSr41 alloys when evaluated using the in vitro direct culture method with BMSCs in a 72 h culture period. The comparison of the mean degradation rates of these Mg alloys with their respective Mg controls, showed that ZSr41A, ZSr41B, and Mg-4Zn-0.5Ca degraded significantly faster. Furthermore, the degradation rates of Mg-xZn-0.5Ca (x = 0.5, 1.0, and 2.0 wt %) were approximately half of ZSr41C and ZSr41D; however, statistical differences were not confirmed for these alloys when compared with their respective Mg controls. Detailed explanations on the degradation mechanism and participation of second phases in the ZSr41 alloys and Mg-xZn-0.5Ca alloys are provided previously.10,11 From this comparison study, the Mg-2Zn0.5Ca alloy stood out as a promising candidate for further testing for skeletal implant applications. 4.2. Cytocompatibility of ZSr41 Alloys in Direct Culture. The direct culture method used in this study showed that BMSCs adhered and remained viable under direct contact at the interface with the ZSr41 alloys; and, the adhesion of BMSCs under indirect contact with the ZSr41 alloys (i.e., adherent to the culture plate surrounding the sample) were unaffected by the solubilized degradation products from the ZSr41 alloys during the 72 h direct culture. Although adherent and viable BMSCs were observed at the cell-ZSr41 interface (Figure 1; Figure 2a, C Kα1 maps) for all four ZSr41 alloys, the significantly lower cell adhesion density on ZSr41A and ZSr41B than the glass reference (Figure 3b, sample) could have been induced by the significantly faster degradation rate of these two alloys (Figure 5). Moreover, the significantly lower BMSC adhesion density at the cell-material interface was most likely caused by the sharper increase in local pH and heterogeneous surface topography (Figure 2a, Mg Kα1 maps) induced by rapid degradation, rather than by the local increase of [Mg2+].11 Our previous study on the effects of pH and supplemented [Mg2+] on BMSC viability and morphology showed that transient media alkalinity played a more important role than [Mg2+] concentration in the media.11 Thus, it was expected that the degradation of ZSr41 alloys in DMEM (which was significantly lower than in EGM-2 and mTeSR1, as discussed in the previous section) and concomitant change in pH and

strength relative to MCDB 131/EGM-2 and mTeSR1. Specific values for osmolality and ionic strength are listed in Table 1. The comparison study showed that buffer capacity, i.e., HCO3− concentration of DMEM, mTeSR1, and MCDB 131/ EGM-2, and to a lesser extent, D-glucose concentration, were likely responsible for the different ranges of degradation rates of the respective ZSr41 alloys and Mg control. Previous in vitro studies showed that the degradation of Mg-based materials was influenced by environmental factors including CO2, buffers, proteins, and other components.18,27,28 Notably, most of these studies focused on evaluating the effects of these environmental factors in incubation media without cells.27,28 For example, Walker et al. demonstrated that the buffer type (NaHCO3 or HEPES) mediated the degradation rates of several Mg-based materials after a 7-day incubation, with a faster rate observed for incubation in HEPES.27 They concluded that for the purpose of degradation testing, incubation in Earle’s balanced salt solution (EBSS) buffered with NaHCO3 provided a close approximation to subcutaneous degradation in a rat model.27 Willumeit et al. utilized artificial neural networks (ANNs) to analyze the correlation between the measured degradation rates (through incubation in the media without cells) and the following parameters, including FBS (0%, 10% and 20% v/v); temperature (20 and 37 °C); CO2 (0%, 2.5% and 5%); O2 (5% and 21%); and media composition, which included glucose (0−4.5 g L−1) and NaCl concentration experimentally, and included NaHCO3, CaCl2, and MgSO4 computationally.28 Following two distinct simulations, they demonstrated that CO2 and NaCl had the strongest influence on the degradation rates of Mgbased metals, followed by both temperature and NaHCO3, and finally by glucose concentration. Interestingly, proteins (i.e., FBS) played the least important role in one scenario but ranked midimportance in the second simulation, which agreed with the experimental discrepancy on the role of proteins.28 Collectively, despite the absence of cells in these studies, the results provided by Walker et al. and Willumeit et al. identified important parameters to consider when using in vitro culture methods to study the degradation rates of Mg-based biomaterials. A close dissection of the factors identified by Willumeit et al. showed that only concentrations of NaCl, NaHCO3, and glucose are relevant to cell-based in vitro culture methods since the O2/ CO2 content (i.e., 95/5%) and temperature (i.e., 37 °C) are fixed in standard cell culture conditions. The cell-based in vitro direct culture method presented the advantages of determining the degradation rates of the Mg-based biomaterials, testing their cytocompatibility, and probing cellular responses at the cell− material interface, all in a single culture system with relevant cells.11 Specifically, in the comparison among the culture systems of BMSC/DMEM, hESC/mTeSR1, and HUVEC/ EGM-2, we demonstrated that [Cl−] was not likely responsible for the different degradation rates of ZSr41 alloys and the Mg control. In contrast, the HCO3− and D-glucose concentrations in the culture media decreased as follows: DMEM < mTeSR1 < MCDB 131/EGM-2, which was directly correlated to the degradation rates of the ZSr41 alloys and Mg controls obtained from the comparison study. Our comparison study highlighted the importance of HCO3− concentration in modulating in vitro degradation of Mg-based materials when using cell-based direct culture methods. Kirkland et al. also suggested that the corrosion of Mg-based materials was mediated by a precipitated calcium phosphate layer which was modulated by HCO3− concentration.29 Further research is needed to verify the individual contributions of HCO3− concentration and DH

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ACS Biomaterials Science & Engineering Mg2+ ion release, would have negligible effects on the viability of BMSCs under indirect contact conditions with the ZSr41 alloys (Figure 3b, plate). The systemic comparison on the cytocompatibility of the ZSr41 alloys with hESCs, HUVECs, and BMSCs showed that the sensitivity of these cells (in their respective culture media) to Mg-based degradation products increased as follows: BMSC < HUVEC < hESC. Previously, we introduced an in vitro cytocompatibility study using H9 hESCs to assess subtle cellular effects (e.g., adhesion, proliferation, and differentiation) induced by the degradation of Mg-based biomaterials,8 because the H9 hESCs showed greater sensitivity to known toxicants for potentially screening subtle cytotoxic effects.34,35 The higher percent viability of BMSCs than hESCs (Figure 7a) confirmed that indeed the viability threshold to the degradation of the ZSr41 alloys and Mg control was much higher for BMSCs than for hESCs; that is, the sensitivity of hESCs was higher to the degradation products of Mg-based biomaterials than BMSCs. Further comparison of the viability of hESCs and HUVECs in the direct culture and under indirect contact with the ZSr41 alloys and Mg control (Figure 7b) confirmed the greater sensitivity of hESCs for the purpose of screening subtle environmental changes. The percent viability of HUVECs in response to the degradation products of ZSr41 alloys and Mg control was higher than the viability of hESCs. In fact, separate experiments on the individual effects of adjusted media alkalinity and Mg2+ ion supplementation on the viability of hESCs, HUVECs, and BMSCs showed that indeed hESC viability was reduced at supplemented [Mg2+] > 10 mM, but HUVECs and BMSCs were not.8,10,11 Additionally, adjusting the culture media to an initial pH of 9.5 followed by a 24-h incubation significantly reduced the viability of HUVECs and BMSCs; however, the same study should be carried out to determine the sensitivity of hESCs to highly alkaline culture media (up to pH 9.5). It was reported that the initial increase of media pH to 8.1 had no adverse effect on hESC proliferation.24 Importantly, the faster degradation rates of the ZSr41 alloys and Mg in hESC/mTeSR1 and HUVECs/EGM-2 than in BMSCs/DMEM culture systems, and the concomitant increase in the concentrations of solubilized degradation products, could also play a role in the percent cell viability when comparing different cell-based culture systems. Since each type of these cells required specific culture media formulations (i.e., mTeSR1 for hESCs, EGM-2 for HUVECs, DMEM for BMSCs) and the respective degradation rates in these media became an intrinsic aspect of utilizing each culture system for studying biomaterials in vitro. In the case of Mg-based biodegradable materials, whose degradation rates depend on the composition of cell culture media, the selection of a relevant in vitro culture system for investigation of their degradation and cytocompatibility becomes a critical issue, which should be carefully considered to narrow the gap of mismatch between in vitro and in vivo results. Collectively, our results not only confirmed the higher sensitivity of hESCs to the degradation products of Mg-based biomaterials, but also emphasized the need of carefully selecting the culture systems for studying Mg-based biomaterials in vitro. Although not statistically significant, our comparison study showed an inverse relationship between mean percent BMSC viability at the cell-material interface and alloy degradation rate (i.e., normalized mass loss rate). Specifically, the percent BMSC viability under direct contact (Figure 7c) followed an inverse trend when compared with the degradation rates of ZSr41 and Mg-xZn-0.5Ca alloys (Figure 6b), suggesting a close relation-

ship between BMSC viability at the cell-material interface and the degradation rate of Mg-based biomaterials (Figure 8). In

Figure 8. Inverse relationship of daily degradation rates of ZSr41 alloys and Mg-xZn-0.5Ca (x = 0.5, 1.0, 2.0, and 4.0 wt %)11 alloys with percent BMSC viability under direct contact at the cell-material interface after 72 h of direct culture in DMEM. Percent BMSC viability is relative to the cells-only positive control. Degradation rates are mean ± SD and percent BMSC viabilities are mean ± standard error of the means; n = 3.

general, the trend showed that the degradation rates of MgxZn-0.5Ca alloys (x = 0.5, 1.0, and 2.0 wt %) allowed for BMSC adhesion and proliferation at the cell-material interface during 72 h of direct culture. In contrast, the faster degrading Mg-4Zn0.5Ca alloy and ZSr41 alloys showed a reduced percent BMSC viability, which could be possibly attributed in part to the significantly faster degradation rates of these alloys.11 In the case of the ZSr41 alloys, the statistically lower viability of BMSCs at the cell−material interface with the ZSr41A and ZSr41B alloys could correlate with the statistically significant faster degradation rates of these two alloys than the Mg control in this study. Since the degradation process of Mg and Mg alloys induces a highly dynamic environment at the cell− material interface, further studies are needed to elucidate the specific roles of each component of the degradation processes on BMSC adhesion and viability.

5. CONCLUSIONS This article reports the behaviors of BMSCs interfacing with four biodegradable Mg-4Zn-xSr alloys (x = 0.15, 0.5, 1.0, or 1.5 wt %; designated as ZSr41A, B, C, and D, respectively). Using the direct culture method, we showed that BMSCs adhered and remained viable under direct contact at the cell-ZSr41 alloy interface; BMSCs under indirect contact with the samples (i.e., adherent to the culture plate surrounding the sample) were unaffected by the solubilized degradation products from the ZSr41 alloys. A systemic comparison study on the culture systems and Mg alloys among ZSr41/BMSC/DMEM, ZSr41/ HUVEC/EGM-2, ZSr41/hESC/mTeSR1, and Mg-xZn-0.5Ca/ BMSC/DMEM, was performed to identify the key parameters to consider when designing in vitro experiments and selecting cell-based direct culture systems for screening Mg-based biomaterials for biomedical implant applications. Our comparison study showed that the in vitro degradation rates of the ZSr41 alloys in distinct culture systems increased as follows: BMSC/DMEM < hESC/mTeSR1 < HUVEC/EGM-2. The differences in in vitro degradation rates were less likely dependent on the following media parameters of chloride ion concentration ([Cl−]), osmolality, and/or ionic strength, but I

DOI: 10.1021/acsbiomaterials.6b00684 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering more likely caused by buffer capacity (i.e., HCO3− concentration), and to a lesser extent, D-glucose concentration. The comprehensive comparison of the cytocompatibility of the ZSr41 alloys and Mg controls in the culture systems of hESC/ mTeSR1, HUVEC/EGM-2, HUVECs, and BMSC/DMEM showed that the sensitivity to Mg-based degradation products for each cell type (in their respective culture media) increased as follows: BMSC < HUVEC < hESC. Moreover, our comparison study showed a general inverse relationship between mean percent BMSC viability at the cell−material interface and alloy degradation rate (i.e., normalized mass loss rate). Overall, the slower degradation rate of Mg-2Zn-0.5Ca alloy in average allowed for higher BMSC adhesion and proliferation at the cell−material interface in 72 h of culture when compared with the ZSr41 alloys, and thus should be further studied as a promising candidate for potential skeletal implant applications.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 951 827 2944. Fax: 951 827 6416. ORCID

Huinan Liu: 0000-0001-9366-6204 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the U.S. National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) of the National Institutes of Health (Award Number 1R03AR069373-01), Hellman Faculty Fellowship (H.L.), and the University of California Regents Faculty Fellowship (H.L.) for financial support. The authors thank U.S. Department of Education for Hispanic Service Institutions Undergraduate Research program (Award Number P031C110131) and California Institute for Regenerative Medicine (CIRM) Bridges to Stem Cell Research program for supporting the undergraduate student researchers (A.L. and A.S., respectively). The authors also thank National Natural Science Foundation of China (Grants 51674077 and 51474063) for financial support. The authors thank the Central Facility for Advanced Microscopy and Microanalysis (CFAMM) at the University of California, Riverside. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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DOI: 10.1021/acsbiomaterials.6b00684 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.6b00684 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX